SUMMARY. The complex spatiotemporal dynamics of messenger RNAs and non-coding RNAs affect virtually all aspects of cellular function. In addition to serving as the central intermediary between DNA and proteins, RNAs regulate gene expression at multiple levels, play roles in epigenetic regulation and genome organization, and serve as physical scaffolds to assemble and integrate macromolecular complexes, with important implications for normal development, as well as disease etiology. Yet, despite the importance of RNA in biology and growing evidence of complex and dynamic localization patterns, robust tools for visualizing RNA molecules in live cells are highly limited. The most widely used RNA tagging system involves addition of 24 MS2 stem loops and binding of 48 molecules of the MS2 binding protein fused to GFP, adding > 1300 nucleotides and > 2.6 MDa to an RNA of interest. While this system has revealed tantalizing glimpses at the individual steps of gene expression regulation, perhaps not surprisingly, it has also been shown to perturb mRNA processing, splicing, localization, and decay. Thus, there is a pressing need for robust, complementary, and minimally perturbing tools to visualize individual RNA molecules in living cells to map the complex and evolving landscape of RNA biology. In this work, we will meet this need by generating a suite of diverse riboswitch-based RNA tags and corresponding fluorescent probes for simultaneous, multi-color imaging of individual RNA molecules in live mammalian cells. Our approach builds on preliminary work from our labs that exploits one of nature?s aptamers, the cobalamin (Cbl)-binding riboswitch as an RNA tag that binds a series of Cbl-linked fluorophores to induce fluorescence turn-on, thus lighting up the RNA of interest. We called this new RNA tagging platform Riboglow and demonstrated its ability to visualize mRNA and small U1 snRNA in live mammalian cells. While the performance of Riboglow was impressive compared to other dye binding aptamers and the gold standard 24xMS2 system, there is significant room for improvement. In this proposal, we will create Riboglow 2.0, with dramatically improved properties by systematically optimizing modules of the RNA/probe platform (Aim 1). In three independent subaims, we will exploit the modular nature of riboswitch structural motifs, the diversity of riboswitch sequences and power of in vitro selection to engineer optimized aptamer-linker pairs, RNA/probe combinations with enhanced fluorescence turn-on, and orthogonal aptamer/probe pairs to enable simultaneous detection of multiple RNAs with spectrally distinct probes. In our second aim, we will create a robust and systematic pipeline for characterizing, validating and benchmarking Riboglow 2.0 (Aim 2). We will define in vitro biochemical and biophysical properties, cellular contrast and single molecule sensitivity, demonstrate functionality for tagging different RNAs in diverse cellular assays, and ensure minimal cytotoxicity and perturbation of RNA function. Integration of Aim 1 and Aim 2 into an iterative cycle of design-engineer- characterize will result in a powerful Riboglow toolbox for diverse biological applications.